Water quality and the CO2-carbonate system during the preconditioning of Pacific oyster (Crassostrea gigas) in a recirculating aquaculture system

The continued increase of the demand for seed of the Pacific oyster (Crassostrea gigas) has driven the aquaculture industry to produce land-based hatcheries using broodstock conditioning. This has led to the need to create closed systems to control the main factors involved in reproduction (temperature and food). Additionally, reproductive synchronization of broodstocks may be considered to ensure homogeneous maturation and spawning among the organisms. In this work, we synchronized the broodstock reproductive stage of Pacific oysters in a recirculating aquaculture system (RAS) using a “preconditioning” process and evaluated the effect of the water quality and the CO2-carbonate system on preconditioned broodstock. The oysters were kept at 12 °C for 45 days in a RAS containing a calcium reactor (C2) or without a calcium reactor (C1, control). Water quality parameters were measured daily, and the oyster’s condition and reproductive development were monitored using condition index, biometrics, and histology, on Days 0, 20, and 45. C1 and C2 systems kept the water quality within the ranges reported as favorable for bivalves. The calcium reactor kept the pH (8.03–8.10), alkalinity (200 mg/L as CaCO3), CO32− (≤ 80 µmol/kg), and Ω aragonite (≤ 1) closer to the ranges reported as optimal for bivalves. However, no significant differences were detected in the total weight and the condition index in C1 and C2. The preconditioning allowed to maintain the organisms in early reproductive development, allowing gametogenesis synchronization to start maturation.

Water quality and the CO 2 -carbonate system during the preconditioning of Pacific oyster (Crassostrea gigas) in a recirculating aquaculture system Salvador Villasuso-Palomares 1 , María T. Gutiérrez-Wing 2 & Carmen G. Paniagua-Chávez 1* The continued increase of the demand for seed of the Pacific oyster (Crassostrea gigas) has driven the aquaculture industry to produce land-based hatcheries using broodstock conditioning. This has led to the need to create closed systems to control the main factors involved in reproduction (temperature and food). Additionally, reproductive synchronization of broodstocks may be considered to ensure homogeneous maturation and spawning among the organisms. In this work, we synchronized the broodstock reproductive stage of Pacific oysters in a recirculating aquaculture system (RAS) using a "preconditioning" process and evaluated the effect of the water quality and the CO 2 -carbonate system on preconditioned broodstock. The oysters were kept at 12 °C for 45 days in a RAS containing a calcium reactor (C2) or without a calcium reactor (C1, control). Water quality parameters were measured daily, and the oyster's condition and reproductive development were monitored using condition index, biometrics, and histology, on Days 0, 20, and 45. C1 and C2 systems kept the water quality within the ranges reported as favorable for bivalves. The calcium reactor kept the pH (8.03-8.10), alkalinity (200 mg/L as CaCO 3 ), CO 3 2− (≤ 80 µmol/kg), and Ω aragonite (≤ 1) closer to the ranges reported as optimal for bivalves. However, no significant differences were detected in the total weight and the condition index in C1 and C2. The preconditioning allowed to maintain the organisms in early reproductive development, allowing gametogenesis synchronization to start maturation.
Pacific oyster (Crassostrea gigas) is the most cultured bivalve worldwide 1 . However, C. gigas production faces a significant challenge: continuous seed production to meet current demands 2 . Consequently, the conditioning of oyster broodstocks in hatcheries to produce high-quality seeds is an important issue. Broodstock conditioning is a process by which an organism's reproductive cycle is controlled by manipulating the main factors involved as the temperature or the feeding rates 3 . However, the onset of the initial reproductive stage in wild broodstocks is variable among organisms, and it is not possible to verify at least the organisms are open. Thus, the reproductive stage of an organism is uncertain at the beginning of conditioning, and the time required to produce mature gametes among the oysters constantly fluctuates 4 . Therefore, the objective of preconditioning is to induce all organisms to reach maturation at the same time.
We define preconditioning as the process of synchronizing a broodstock's reproductive stage by manipulating the biological zero temperature (temperature below which reproductive development does not occur 3 ) to initiate maturation at the same reproductive stage. The maintenance of a preconditioned broodstock will allows for staggered larval production throughout the year. The needs of oyster farmers can be better served with a continuous, timely supply of high-quality larvae.
Recirculating aquaculture systems (RAS). The experimental system for each condition (C1 and C2) consisted of one RAS with a culture tank containing 1.3 m 3 seawater. A moving bed biofilter, which contained 110 L of Kaldnes™ K1 media (specific surface area: 500 m 2 /m 3 ) to support nitrifying bacteria, was used for nitrification and degassing. A 100-L plastic container with 20 L of an elliptical polyethylene floating filter consisting of bead media AB1 (Pentair Aquatic Eco-System) was used for solid waste removal. The water was circulated at a rate of 57 L/min using a magnetic pump (Little Giant Model 3-MD-SC, Oklahoma City). The water temperature was controlled with an air-cooled heat pump (Delta Star ® , Aqua Logic DSHP-5) and was adjusted to 12 °C before replacement to avoid thermal shock in the organisms. A weekly 500-L water replacement was performed on each RAS to remove solids.
Calcium reactor (CA). A calcium reactor was used to control the CO 2 -carbonate system. The calcium reactor's purpose was to constantly add CO 3 2− ions to buffer H + produced from nitrification and CO 2 hydrolysis. For Condition C2, the calcium reactor was connected to the biofilter. The calcium reactor consisted of a reaction chamber (40 cm high × 10.16 cm diameter) connected by a hose (0.635 cm diameter) to a counter bubble chamber (16.5 cm high × 5.2 cm diameter), and a Quiet One ® 1200 magnetic pump (Lifergard Aquatics) was used for water circulation. The reaction chamber contained 1 kg of a crushed coral-based calcium reactor medium (ReBorn™, Florida, US). Coral-based media dissociation was increased by CO 2 (1.3-1.5 mL/min) injection through the counter bubble chamber. The calcium reactor design was described by Sanjay Joshi 10 .
Oyster preconditioning. Once the organisms were distributed across the two experimental conditions, the oysters were kept below their biological zero condition (12 °C) for 45 days and fed daily with 20 mL (2 × 10 9 cells/ mL) of microalgae concentrate (Shellfish diet 1800™, Reed Mariculture). This commercial concentrate contained a mixture of six different microalgae: Isochrysis sp., Pavlova sp., Tetraselmis sp., Chaetoceros calcitrans, Thalassiosira weissflogii, and Thalassiosira pseudonana. The final microalgae concentration per RAS was 27 × 10 3 cells/mL. Twenty oysters were sampled on Days 20 and 45 to determine their total weight, condition index, and reproductive development.
Water quality. Water quality was measured daily for both experimental conditions. Temperature, salinity, and dissolved oxygen (DO) were measured with a Pro Dss multiparameter meter (YSI ® ; Ohio, US). The total ammonia nitrogen (TAN) was determined using Solorzano's indophenol method 11 . Nitrite nitrogen (NO 2 -N) was determined with a method described by Shin and modified by Bendschneider and Robinson 12 for seawater. Nitrite nitrogen was measured using sulfanilamide hydrochloride and N-(1-naphthy1)-ethylenediamine dihydrochloride to form an azo dye, which was measured at a wavelength of 543 nm. Nitrate nitrogen (NO 3 -N) was determined by an ultraviolet spectrophotometric screening method according to a methodology described in Standard Methods for the examination of water and wastewater 13 . TAN, NO 2 -N, and NO 3 -N measurements were made using an Epoch™ spectrophotometer (Biotek ® Instruments, Winooski, US). All nitrogen compound measurements were carried out in triplicate. ) concentration, and the saturation states of calcite (Ω ca ) and aragonite (Ω ar ) were calculated according to measured values of pH, alkalinity, salinity, and temperature using the dissociation constants described by Mehrbach et al. 15 with a software CO 2 System (CO 2 sys) by Lewis and Wallace 16 .
Survival, total weight, and condition index (CI). Survival was monitored throughout the experiment, and the survival rate under both the C1 and C2 experimental conditions was recorded. The same oysters were used to measure the total weight and determine the condition index (CI). The CI was calculated according to an equation described by Hickman and Illingworth 17 Reproductive development. Tissue samples (4-mm wide slices) obtained from a location between the labial palps and gills of the 20 oysters in each sample were fixed in Davidson's solution for 48 h 18 . After fixation, the samples were dehydrated in ascending concentrations of ethanol solutions and embedded in paraffin wax. Then, 5-µm thick slices were stained with hematoxylin and eosin and mounted on glass slides 18 . The slides were observed with an Olympus microscope (CX31RTSF) at 400 ×, and reproductive development was determined using the seven-stage reproductive scale (undifferentiated, developing -early active, developing-late active, ripe, partially spent, totally spent, postspawning, and resorption) described by Steele and Mulcahy 19 .
Data analysis. The total weight and CI were tested using a Mann-Whitney U test on Days 20 and 45 for C1 and C2. A value of P < 0.05 was chosen as the level of significance. Statistical analysis was performed using Statistica 7.1 (Stat Soft, Inc.).
Ethical approval and consent to participate. No approval from research ethics committees was required because the experimental work was conducted with an unregulated invertebrate species.

Results
Water quality. The temperatures for C1 and C2 were maintained at biological zero (12 °C) during experimentation. The mean temperature during the 45 days of experimentation was 11.34 °C ± 0.39 °C for C1 and 11.56 °C ± 0.55 °C for C2. The mean DO concentrations under both experimental conditions were 8.00 mg/L ± 0.10 in C1 and 7.99 mg/L ± 0.14 in C2. The salinity was 34.02 ± 0.08 ppt in C1 and 33.81 ± 0.07 ppt in C2 (Table 1).
After 45 days of experimentation, the mean pCO 2 was 982 ± 191 µatm for C1 and 1030 ± 185 µatm for C2. The mean HCO 3 − concentration in C2 (2630 ± 304 µmol/kg) was higher compared with C1 (1942 ± 104 µmol/kg). The CO 3 2− concentration of 98 ± 13 µmol/kg for C2 was higher compared with the concentration of 57 ± 7 µmol/kg for C1. The Ω ar was < 1 in C1 and > 1 in C2. The Ω ca was > 1 under both experimental conditions. Nevertheless, the mean concentration of Ω ca after 45 days of experimentation was 2.36 ± 0.32 for C2 and 1.36 ± 0.17 for C1 (Table 2). Survival, total weight, and condition index (CI). The total survival after 45 days of experimentation was 83.21% for C1 and 82% for C2. The total individual weight was 46.2 ± 3.4 g at the beginning of the experi-CI = dry weight of the soft tissue total weight − shell weight × 100. www.nature.com/scientificreports/ ment (Day 0). At the end of the experiment, the total individual weight for oysters in C1 was 46.8 ± 8.5 g and 47.6 ± 7.4 g in C2 (Fig. 1A). No significant differences were detected in the total weight on Day 20 (P = 0.28) or on Day 45 (P = 0.82) between C1 and C2. The oysters in C1 were significantly heavier (P = 0.28) on Day 20 than on Day 45. For C2, no significant differences (P = 0.40) were detected in the total weight between Days 20 and 45. No significant differences were detected in the total weight between Days 0 and 45 in oysters from C1 (P = 0.93) and C2 (P = 0.93). The condition index on Day 0 was 6.3 ± 2.99 and reached 4.1 ± 1.05 in C1 and 4.7 ± 0.98 in C2 (Fig. 1B). Significant differences were detected in CI on Day 20 between C1 and C2 (P = 0.041). No significant differences were detected in CI on Day 45 between C1 and C2 (P = 0.15).

Reproductive development.
Males and females were observed in all samples except on Day 20 for C2 ( Fig. 2A,B). At the beginning of the experiment, 100% of the females and 20% of the males were in the resorption stage. Additionally, 60% of the males were in the developing-early active stage, and only 20% of the males were ripe (Fig. 2C,D). For Days 20 and 45, all females were in the resorption stage, and all the males were in the developing-early active stage for both experimental conditions (Fig. 3A,B).

Discussion
We define preconditioning as a process that synchronizes the reproductive stage of an oyster's previous broodstock conditioning. Preconditioning could become a helpful process for achieving predictable conditioning times. To achieve successful preconditioning, it is necessary to keep the temperature at biological zero to avoid initiating the oyster reproductive cycles. Additionally, it is necessary to keep the water quality and CO 2 -carbonate system at favorable ranges to avoid long-term exposure to stress-inducing conditions. The exposition of bivalves to unfavorable environmental conditions during their reproductive cycle could promote the lysis of gametes as a strategy to recycle energy to satisfy the increase in basal metabolism 19 .
The temperature (< 12 °C), salinity (~ 34 ppt), and OD (> 7.0 mg/L) after 45 days of experimentation were kept within optimal ranges to allow for reproductive development delay 20,21 . Additionally, food supply was not a limiting factor since it was maintained at a recommended concentration (100 × 10 6 cells per oyster per day) to ensure proper nutrition of C. gigas at low temperatures and outside the reproductive cycle 22 .  www.nature.com/scientificreports/ The TAN concentration under both experimental conditions was in the ranges reported by Buchanan et al. 20 (0.10 mg/L and 4.2 mg/L) for oyster conditioning in an RAS for eight weeks, with no negative effects. Under our experimental conditions, the nitrite nitrogen concentration was within the range recommended by Stone and Thomforde 23 for aquatic organisms (0.05-0.5 mg/L). Therefore, we can conclude that the concentrations of TAN and N-NO 2 under both experimental conditions (C1 and C2) did not significantly affect the oysters.
A controlled CO 2 -carbonate system is essential for oyster preconditioning in RASs because of the complex interactions of alkalinity, pH, pCO 2 , CO 3 2− , Ω ar , and Ω ca , with the nitrifying bacteria from the biofilter and with the oysters. During the nitrification process, nitrifying bacteria consume HCO 3 − as an inorganic carbon source and produce H + as a metabolic byproduct 6 . The constant addition of H + to the water and consumption of HCO 3 − reduces the pH and alkalinity, which consists of 90% HCO 3 − , promoting water acidification. However, CO 2 accumulation can also affect pH and alkalinity.
When CO 2 interacts with water, it produces carbonic acid (H 2 CO 3 ), which decreases pH and alkalinity. H 2 CO 3 can dissociate in H + and HCO 3 − or interact with CO 3 2− to produce 2HCO 3 − . Then, H 2 CO 3 reduces the concentration of CO 3 2and the saturation state of Ω ar and Ω ca 24 . Therefore, the increase in H + from nitrification and CO 2 accumulation reduces the buffer capacity of the water, promoting corrosive conditions for the oysters. Long-term exposure to corrosive conditions can negatively affect the acid-base balance, somatic growth, and condition index, which increases shell dissolution, energy requirements, and mortality 25 .
Alkalinity determines the acid-neutralizing capacity of water. For C1, the alkalinity was lower since C1 did not have an external source of CO 3 2− , while C2 had a calcium reactor. For RAS, an alkalinity of 200 mg/L CaCO 3 is recommended for the proper operation of biofilters 26 . However, the low alkalinity did not impact biofiltration performance, as the nitrogen compound concentrations were kept within the optimal ranges for both experimental conditions.
The pH obtained under both experimental conditions was < 8.0 due to the nitrification process and due to the hydrolysis of CO 2 6,7 . The pH in C1 and C2 differed from those reported to be optimum for C. gigas, between 8.03 and 8.10 8,23 . Nevertheless, Boulais et al. 27 tested a pH of 7.5 on the gametogenesis of C. virginica and did not find any significant effects. Similarly, Lannig et al. 28 did not find significant differences in the standard metabolic rates at a pH of 8.07 and at a pH of 7.68 at 15 °C for C. gigas. Therefore, in our study, the pCO 2 was not high enough and the pH was not low enough to negatively affect the oysters.
In our experiment, the pCO 2 under both experimental conditions was higher than those reported as normal conditions for bivalves (374-658 µatm) but lower than values at which adverse effects have been reported (2170-2625 µatm) 8,28-32 .
The CO 3 2− concentration for C1 was below the threshold of 80 μmol/kg, which can promote a drastic decline in calcification 7 . Additionally, Ω ar was > 1, and Ω ca was slightly higher than 1, which could affect the calcification rates and shell dissolution rates to compensate for the saturation state of Ω ar and Ω ca 33 . According to our results, a calcium reactor helps maintain pH, alkalinity, HCO 3 − , CO 3 2− , Ω ar , and Ω ca at values close to the optimal ranges. However, no significant differences were detected in total weight or CI between C1 and C2 after 45 days of experimentation.
The water quality factors, alkalinity, pH, and pCO 2 under both experimental conditions were within ranges that are reported to be favorable to C. gigas, so the RASs probed to be reliable providing optimal conditions for the preconditioning. Despite the relatively low CO 3 2− concentration and Ω ar saturation state in C1, no differences were observed in the CI or the total weight of the oysters after 45 days of experimentation. Therefore, using a calcium reactor may not be necessary during preconditioning with the loading and times tested.
The temperature is the most important factor involved in the reproductive cycle of C. gigas because can accelerate or delay reproductive development 4 , so it was vital to maintain the 12 °C during preconditioning to avoid the initiation of gametes maturation. For C. gigas has been reported when organisms are maintained below the minimum spawning temperature, the resorption process could initiate, which is characterized by the cleaning of the mature gametes so that the gonad can prepare for its next reproductive cycle 19 . Thus, the preconditioning temperature allowed male oysters in resorption and ripe stages could change to an early reproductive development stage. In addition, several authors have reported a minimum spawning temperature of ~ 18 °C for C. gigas 19,34 . The temperatures in C1 and C2 were never higher than 12 °C. Therefore, we can conclude that gametogenesis synchronization of C. gigas under both experimental conditions was due to the preconditioning process and not due to the complete or partial spawning of the oysters.
The results for oyster reproductive development under both experimental conditions showed that 12 °C is a favorable temperature for C. gigas to allow for gametogenesis synchronization and is low enough to avoid gamete maturation. Additionally, the results showed that 45 days of preconditioning could be enough to promote gametogenesis synchronization in C. gigas. Also, the results showed that the calcium reactor has no significant effect on the gametogenesis synchronization in C. gigas. However, further investigations are necessary to evaluate gametogenesis synchronization for longer times, if preconditioning enhances the maturation process during broodstock conditioning and the effect of preconditioning in more than one reproductive cycle.

Data availability
The datasets generated in the current study are available from the corresponding author upon reasonable request.